Interactions between lymphotoxin (LT)α1β2 on inducer cells and the lymphotoxin β receptor (LTβR) on stromal cells initiate development of lymph nodes and Peyer’s patches. In this study, we assessed the contributions of LTα and LTβR to the development of cryptopatches (CP), aggregates of T cell precursors in the mouse small intestine. Mice genetically deficient in LTα or LTβR lacked CP. Bone marrow from LTα-deficient mice was unable to initiate development of CP or isolated lymphoid follicles (ILF) after transfer to CD132-null mice lacking CP and ILF. However, LTα-deficient bone marrow-derived cells contributed to CP formed in CD132-null mice receiving a mixture of wild-type and LTα-deficient bone marrow cells. Transfer of wild-type bone marrow into irradiated LTα-deficient mice resulted in reconstitution of both CP and ILF. However, the LT-dependent formation of CP was distinguished from the LT-dependent formation of ILF and Peyer’s patches by not requiring the presence of an intact NF-κB-inducing kinase gene. CP but not ILF were present in the small intestine from NF-κB-inducing kinase-deficient alymphoplasia mice, indicating that the alternate NF-κB activation pathway required for other types of LTβR-dependent lymphoid organogenesis is dispensable for CP development. In addition, we identified VCAM-1+ cells within both CP and ILF that are candidates for the stromal cells involved in receiving LT-dependent signals from the hemopoietic precursors recruited to CP. These findings demonstrate that interactions between cells expressing LTα1β2 and LTβR are a shared feature in the development of all small intestinal lymphoid aggregates.

Cryptopatches (CP)3 are aggregates of immature T cell precursors that develop postnatally and are found adjacent to crypts in the mouse small intestine (1). These T cell precursors are c-kit+, IL-7Rα+, and Thy-1+ and are in close contact with CD11c+ dendritic cells concentrated at the periphery of the CP (1). CP are regarded as a site where extrathymic differentiation of T cells can take place, as transfer studies have demonstrated that T cell precursors isolated from CP can differentiate into mature T cells expressing αβ or γδ TCRs that reside in the intestinal epithelium (2). CP and intestinal intraepithelial lymphocytes (IEL) can also be reconstituted with wild-type bone marrow (BM) in athymic IL-2R common γ-chain-deficient double mutant mice that genetically lack the thymus, CP, and IEL (3). Furthermore, athymic nu/nu mice have a normal number of CP and also have functional IEL (1). The initial cellular interactions required for the generation of CP have not been fully defined. A second type of lymphoid aggregate that develops postnatally in the mouse small intestine is the isolated lymphoid follicle (ILF) (4). An ILF is a single follicle primarily composed of mature B lymphocytes that are covered by an epithelial layer that includes specialized M cells similar to those found in the follicle-associated epithelium overlying Peyer’s patches (PP) (4).

Interactions between inducer cells expressing the lymphotoxin (LT)α1β2 heterotrimer and stromal cells expressing LT β receptor (LTβR) are required to initiate the development of secondary lymphoid tissues including peripheral lymph nodes and PP (5, 6, 7). Analysis of a series of knockout mouse models has demonstrated that the LTβR signaling pathway involved in lymphoid organogenesis involves NF-κB-inducing kinase (NIK), IκB kinase (IKK) α, NF-κB2 (p100), and RelB (8, 9, 10, 11, 12). This alternate or noncanonical pathway of NF-κB activation is distinct from the classical pathway of NF-κB activation initiated by assembly of an IKK complex containing IKKα, IKKβ, and IKKγ (13). Previous studies have shown that ILF are absent in LTα-deficient mice and LTβR-deficient mice (4, 14). BM chimera experiments demonstrated that ILF development requires LTα expression by the donor hemopoietic cells and LTβR expression by the host cells (14). These findings suggest that the cellular interactions required to initiate ILF development are similar to those involved in PP and lymph node development. Not all organized lymphoid structures require LTα to initiate development. The nasal-associated lymphoid tissue (NALT) can form in LTα-deficient mice, although the NALT of LTα-deficient mice has a paucity of lymphocytes compared with wild-type NALT (15, 16).

Less is known about the involvement of LTα and LTβR in the development of CP. The presence of CP has been reported in both LTα-deficient mice and aly/aly mice that are deficient in NIK (1, 4). To better define the contribution of LTα and LTβR to CP development, we conducted a quantitative analysis of the frequency of CP and ILF in LTα- and LTβR-deficient mice and determined if precursors in the BM of LTα-deficient mice could reconstitute the development of CP in IL-2R common γ-chain-deficient mice (CD132-null mice) that lack CP. We find that CP development requires the presence of both LTα and LTβR, but does not require the presence of a functional NIK, unlike other LTβR-dependent lymphoid organogenesis. We also have identified the presence of VCAM-1+ stromal cells at the periphery of both CP and ILF and suggest that these cells are important targets for LTα effects during development of these intestinal lymphoid aggregates.

CD132-null mice (B6.129S4-IL2rgtm1Wjl/J) (17), LTα−/− mice(B6.129S2-Ltatm1Dch/J) (5), and TNFRI/TNFRII−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Breeding colonies of the CD132-null and LTα−/− mice were maintained in a conventional mouse facility at Emory University. A few of the LTα−/− mice examined in the course of this study were bred in a colony established by Thomas W. Spahn and Torsten Kucharzik at the University of Münster (Münster, Germany). The LTα−/− mice used for histological analysis ranged from 6 wk to 5 mo in age. Mutant aly/aly mice were obtained from a colony maintained by K. Newell at Emory University. C57BL/6.SJLCD45.1 mice were purchased from Taconic (Germantown, NY). Formalin-fixed small intestinal tissue from 3-mo-old LTβR−/− mice (18) was kindly provided by Yang-Xin Fu (University of Chicago, Chicago, IL).

The mAbs used to stain cell suspensions for flow cytometry included mAbs to CD3, CD19, CD45.1, and CD45.2. The mAbs purchased from BD PharMingen (San Diego, CA) were biotin-anti-CD3 (145-2C11) and biotin-anti-CD19 (1D3). The mAbs purchased from eBioscience (San Diego, CA) were FITC-anti-CD45.2 (104) and APC-anti-CD45.1 (A20). Binding of biotinylated mAb was detected with streptavidin conjugated to allophycocyanin (Caltag Laboratories, Burlingame, CA). The mAbs used for immunofluorescence included mAbs to c-kit (CD117), B220, CD19, Thy-1.2, CD11c, CD45.1, CD45.2, PECAM-1 (CD31), VCAM-1 (CD106), and MAdCAM-1. The mAbs purchased from BD Pharmingen were PE-anti-B220 (RA3-6B2), APC-anti-B220 (RA3-6B2), PE-anti-Thy-1.2 (53-2.1), FITC-anti-Thy-1.2 (53-2.1), PE-anti-CD11c (HL3), PE-anti-CD31 (MEC13.3), and purified anti-VCAM-1 (429). Biotin-anti-c-kit (2B8) and Alexa Fluor 647-anti-c-kit (2B8) were purchased from Caltag. Abs purchased from eBioscience included FITC-anti-CD45.1 (A20), APC-anti-CD45.1 (A20), PE-anti-CD45.2 (104), biotin-anti-VCAM-1 (429), and biotin-anti-MAdCAM-1 (MECA-367). Splenic germinal centers were detected using HRP-conjugated peanut agglutinin (Sigma-Aldrich, St. Louis, MO).

BM was eluted from the tibia and femur of donor mice. A single-cell suspension was prepared in complete media and depleted of mature T cells using CD90 (Thy-1) MACS beads and an LD depletion column (Miltenyi Biotec, Auburn, CA). T cell depletion was verified by flow cytometry. CD132-null recipient mice were given 600 rad of gamma-irradiation from a cesium source. LTα−/− recipient mice were given a total of 1000 rad. Donor BM cells were resuspended in PBS and 2 × 107 cells were injected i.v. into recipient mice. For competitive BM reconstitution experiments, C57BL/6.SJLCD45.1 and LTα−/− BM were mixed 1:1 and injected into irradiated CD132-null recipients. Mice were given drinking water supplemented with neomycin sulfate (2 mg/ml) for 2 wk after transfer.

Peripheral blood chimerism was monitored at several time points following transfer by flow cytometric analysis of PBMC isolated through centrifugation over Histopaque-1077 (Sigma Diagnostics, St. Louis, MO). To block nonspecific FcR-mediated binding, the isolated PBMC were incubated with supernatant from the 2.4G2 hybridoma line (American Type Culture Collection, Manassas, VA) for 10 min before addition of mAb. After staining with mAb to CD45.1, CD45.2, CD3, and CD19, the cells were analyzed on a FACSCalibur cytometer (BD Biosciences) using CellQuest software (version 3.3).

Small intestines were cleaned, opened longitudinally, and flushed with cold PBS. Small sheets of tissue (∼15 × 20 mm) were stacked (three at a time) and covered with OCT freezing medium. Blocks were quick-frozen in cold isopentane on dry ice. Frozen sections of 6-μm thickness were cut with a cryostat, air dried, and fixed for 10 min in cold acetone. Endogenous peroxidase activity was quenched with 0.3% H2O2 in PBS for 30 min at 37°C. Sections were washed in PBS and blocked in TNB buffer (PerkinElmer Life Sciences, Boston, MA). Primary Ab (biotinylated mAb to c-kit, CD19, VCAM-1, and MAdCAM-1) diluted in TNB buffer was applied overnight at 4°C. Primary Ab was detected using a streptavidin-HRP conjugate followed by FITC or Cy5-conjugated tyramide from a tyramide signal amplification (TSA) kit (PerkinElmer Life Sciences). In some cases, VCAM-1 was detected using a purified primary mAb followed by biotinylated polyclonal goat-anti-rat Ig (BD PharMingen), streptavidin-HRP, and FITC-tyramide. To detect splenic germinal centers, HRP-conjugated peanut agglutinin was used as a primary reagent followed by TSA amplification. TSA amplification was followed by direct detection of surface markers using anti-B220, anti-Thy-1.2, anti-CD11c, or anti-CD31. For experiments where TSA amplification was not employed, acetone-fixed sections were blocked with TNB buffer and stained with directly labeled Abs (anti-CD45.1, -CD45.2, -B220, or -c-kit). Images were acquired using a Zeiss LSM510 confocal microscope.

Frozen sections of small intestine were fixed in ethanol and stained with H&E (Sigma-Aldrich). The total crypt area of each slide was calculated using Scion Image analysis software (Scion, Frederick, MD). Lymphoid aggregates were counted, and their density was expressed as the number of lymphoid aggregates per square centimeter of small intestinal crypt area. The entire length of each small intestine was examined, with special attention to distal portions.

Given that LTα1β2-LTβR interactions are crucial for the development of lymph nodes, PP, and ILF, we examined LTα−/− small intestinal tissue for the presence of CP. Horizontal frozen sections of small intestine from LTα−/− mice were stained with H&E or immunostained with Abs to c-kit, a marker expressed by T lymphoid precursor cells in CP. No lymphoid aggregates were found on H&E-stained sections, indicating CP (and ILF, as reported previously) (14) were absent from these animals (Fig. 1). Immunostaining with anti-c-kit confirmed the absence of c-kit+ clusters indicative of CP in intestinal crypt areas. In addition, lymphoid aggregates were absent from H&E-stained sections of small intestine from LTβR−/− mice (Fig. 2,A). This observation indicates that CP development is specifically dependent on membrane-bound LTα1β2 interactions with LTβR, and the contribution of the secreted lymphotoxin homotrimer (LTα3), which binds to TNF receptors, is dispensable. Furthermore, small intestines of mice deficient in both TNFRI and TNFRII contain normal CP (data not shown). In agreement with a previous report (4), the small intestine of aly/aly mice was found to contain normal c-kit+ CP but showed no trace of ILF (Fig. 2 B).

FIGURE 1.

CP are absent in LTα-deficient mice. Horizontal sections of the small intestine from age-matched C57BL/6 (A) and LTα−/− mice (B) were stained with H&E or with anti-c-kit mAb and examined by immunofluorescence and confocal microscopy. Scale bar, 2 mm; inset, 100 μm. Red circles indicate lymphoid aggregates.

FIGURE 1.

CP are absent in LTα-deficient mice. Horizontal sections of the small intestine from age-matched C57BL/6 (A) and LTα−/− mice (B) were stained with H&E or with anti-c-kit mAb and examined by immunofluorescence and confocal microscopy. Scale bar, 2 mm; inset, 100 μm. Red circles indicate lymphoid aggregates.

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FIGURE 2.

CP are absent in LTβR-deficient mice, but are present in aly/aly mice. Formalin-fixed, paraffin-embedded sections of small intestine from LTβR−/− mice (A) were stained with H&E. Horizontal frozen sections of small intestine from aly/aly mice (B) were stained with H&E or immunostained with mAbs and examined by confocal microscopy. Scale bar in A and B, 1 mm; B inset, 20 μm. Red circles indicate lymphoid aggregates.

FIGURE 2.

CP are absent in LTβR-deficient mice, but are present in aly/aly mice. Formalin-fixed, paraffin-embedded sections of small intestine from LTβR−/− mice (A) were stained with H&E. Horizontal frozen sections of small intestine from aly/aly mice (B) were stained with H&E or immunostained with mAbs and examined by confocal microscopy. Scale bar in A and B, 1 mm; B inset, 20 μm. Red circles indicate lymphoid aggregates.

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The absence of CP in LTα−/− mice suggested a role for LT in CP development. To test this hypothesis, we generated BM chimeras in which irradiated CD45.2+ CD132-null mice were given T cell-depleted BM i.v. from CD45.1+ wild-type or CD45.2+ LTα−/− congenic animals. CD132-null mice, which do not have peripheral lymph nodes or PP and have only a small number of IEL, have been previously reported to be a good recipient strain for analysis of lymphoid aggregate reconstitution in the mouse small intestine (3). CD45 allele analysis of peripheral blood leukocytes by flow cytometry showed that >97% of CD3+ T lymphocytes and >98% of CD19+ B lymphocytes were of donor origin 6 wk after BM transfer (data not shown). At 10 wk posttransfer, frozen sections of small intestine stained with H&E revealed numerous lymphoid aggregates (33 per square centimeter of intestinal crypt area) in CD132-null animals that received wild-type BM (Fig. 3). However, the density of lymphoid aggregates in CD132-null animals that received LTα−/− BM was drastically reduced to 1.4 per square centimeter. Anti-c-kit immunostaining identified CP composed of donor-derived cells in wild-type-reconstituted animals. However, only rare small c-kit+ CP were identified in CD132-null mice that received LTα−/− BM. Likewise, immunostaining of spleen sections revealed normal white pulp T and B cell areas containing germinal centers in wild-type BM-reconstituted CD132 animals. Spleen sections from LTα−/− BM-reconstituted animals, by contrast, contained small, disorganized white pulp areas without germinal centers. This splenic phenotype closely resembles that of LTα−/− mice. These results confirmed that in the CD132-null mice with an intestinal environment that is highly permissive for lymphoid aggregate reconstitution, BM-derived lymphoid precursor cells that lack LTα cannot induce normal CP or ILF formation.

FIGURE 3.

Wild-type BM, but not LTα-deficient BM, can reconstitute CP and ILF in CD132-null mice. Horizontal frozen sections of small intestine from CD132-null mice reconstituted with wild-type BM (n = 3) (A) or LTα−/− BM (n = 4) (B) were stained with H&E or anti-c-kit and anti-B220 mAbs and examined by immunofluorescence and confocal microscopy. Quantitation of reconstituted lymphoid aggregates (C) was done by counting aggregates on H&E-stained sections and dividing by the total crypt area analyzed using image analysis software. A minimum of 2 cm2 of intestinal crypt area was analyzed. Spleen sections from wild-type BM-reconstituted (D) and LTα−/− BM-reconstituted (E) CD132-null mice were stained with HRP-conjugated peanut agglutinin followed by TSA and anti-Thy-1.2 and anti-B220 mAbs. Scale bars in A and B, 2 mm; B inset, 50 μm; A inset, D, and E, 100 μm.

FIGURE 3.

Wild-type BM, but not LTα-deficient BM, can reconstitute CP and ILF in CD132-null mice. Horizontal frozen sections of small intestine from CD132-null mice reconstituted with wild-type BM (n = 3) (A) or LTα−/− BM (n = 4) (B) were stained with H&E or anti-c-kit and anti-B220 mAbs and examined by immunofluorescence and confocal microscopy. Quantitation of reconstituted lymphoid aggregates (C) was done by counting aggregates on H&E-stained sections and dividing by the total crypt area analyzed using image analysis software. A minimum of 2 cm2 of intestinal crypt area was analyzed. Spleen sections from wild-type BM-reconstituted (D) and LTα−/− BM-reconstituted (E) CD132-null mice were stained with HRP-conjugated peanut agglutinin followed by TSA and anti-Thy-1.2 and anti-B220 mAbs. Scale bars in A and B, 2 mm; B inset, 50 μm; A inset, D, and E, 100 μm.

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The results from the CD132-null BM reconstitution experiments described above predicted that LTα-expressing precursor cells from wild-type BM would be able to reconstitute CP and ILF formation in LTα−/− mice, which retain LTβR expression on radioresistant stromal cells. Reconstitution of ILF in this type of BM chimera experiment was previously reported (14). T cell-depleted BM from CD45.1+ wild-type B6 mice was injected i.v. into irradiated CD45.2+ LTα−/− mice. Peripheral blood leukocyte chimerism was >88% donor origin for T cells and >99% for B cells by 6 wk posttransfer (data not shown). Horizontal frozen sections of small intestine were examined for the presence of CP and ILF 11 wk after reconstitution. Indeed, lymphoid aggregates could be identified in these chimeric animals by H&E staining (Fig. 4). Immunofluorescence staining for c-kit and B220 identified the presence of both CP and ILF.

FIGURE 4.

Wild-type BM can reconstitute CP in LTα−/− mice. Horizontal frozen sections of small intestine from LTα−/− mice that received wild-type BM (n = 4) were stained with H&E (A) or with immunofluorescent Ab combinations as indicated B and C. c-kit+B220 CP (B) were identified, as well as B220+ ILF (C) as previously shown. Scale bar in A, 2 mm; B and C, 20 μm. Red circles indicate lymphoid aggregates.

FIGURE 4.

Wild-type BM can reconstitute CP in LTα−/− mice. Horizontal frozen sections of small intestine from LTα−/− mice that received wild-type BM (n = 4) were stained with H&E (A) or with immunofluorescent Ab combinations as indicated B and C. c-kit+B220 CP (B) were identified, as well as B220+ ILF (C) as previously shown. Scale bar in A, 2 mm; B and C, 20 μm. Red circles indicate lymphoid aggregates.

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Given our results that BM-derived lymphoid progenitor cells that lack LTα cannot induce CP or ILF formation in irradiated CD132-null mice, we wondered whether these cells could contribute to CP and ILF induced by LTα-competent progenitors. To answer this question, a competitive BM experiment was done using irradiated CD132-null recipients and a 1:1 mixture of T cell-depleted CD45.1+ wild-type BM and CD45.2+ LTα−/− BM. Lymphoid aggregates (31 per square centimeter of intestinal crypt area) were identified in the small intestines of these chimeras by H&E staining of horizontal frozen sections. Parallel sections to those stained with H&E were immunostained with anti-B220 to identify ILF or anti-c-kit to identify CP in combination with anti-CD45.1 and anti-CD45.2. Immunofluorescence and confocal microscopy identified the presence, in roughly equal proportions, of CD45.1+ (wild-type origin) and CD45.2+ (LTα−/− origin) cells in both ILF and CP (Fig. 5). This result indicates that LTα−/− BM-derived precursor cells, although incapable of initiating CP and ILF development, can contribute to lymphoid structures formed by wild-type BM-derived cells.

FIGURE 5.

Lymphoid precursor cells derived from LTα−/− BM can contribute to CP and ILF initially developed by wild-type BM-derived precursor cells in mixed BM reconstitution experiments. Irradiated CD132-null mice (n = 4) were given a 1:1 mixture of wild-type BM and LTα−/− BM. Horizontal frozen sections of small intestine were stained with Abs against CD45.1, defining cells derived from wild-type BM, and CD45.2, defining cells derived from LTα−/− BM, in the indicated color combinations. B220 signal defining isolated lymphoid follicles (A) and c-kit signal defining cryptopatches (B) were grayed for clarity in illustrating the relative contributions of CD45.1 and CD45.2.

FIGURE 5.

Lymphoid precursor cells derived from LTα−/− BM can contribute to CP and ILF initially developed by wild-type BM-derived precursor cells in mixed BM reconstitution experiments. Irradiated CD132-null mice (n = 4) were given a 1:1 mixture of wild-type BM and LTα−/− BM. Horizontal frozen sections of small intestine were stained with Abs against CD45.1, defining cells derived from wild-type BM, and CD45.2, defining cells derived from LTα−/− BM, in the indicated color combinations. B220 signal defining isolated lymphoid follicles (A) and c-kit signal defining cryptopatches (B) were grayed for clarity in illustrating the relative contributions of CD45.1 and CD45.2.

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The earliest recognizable step in PP organogenesis is the formation of clusters of VCAM-1+ stromal cells at embryonic day 15.5 (19). These VCAM-1+ clusters, which express LTβR, are colonized by LTα-expressing CD3-CD4+IL-7Rα+ inducer cells at around E16.5-E17.0 (19, 20). LTα−/− and wild-type mice treated with LTβRIg fusion protein during embryonic development do not form these VCAM-1+ organizing centers and completely lack PP (5, 6). Therefore, we sought to determine whether VCAM-1+ stromal cells similar to those crucial to PP development were present in CP and ILF in wild-type animals. Immunostaining with anti-VCAM-1 Abs followed by tyramide signaling amplification revealed the presence of VCAM-1+ cells around the periphery of wild-type CP (Fig. 6). CP and ILF from BM-reconstituted CD132-null animals also contained VCAM-1+ cells. These VCAM-1+ cells did not colocalize with endothelial markers such as CD31 or MAdCAM-1, indicating that they are not endothelial cells. They also did not colocalize with markers of the other known cell types in these lymphoid structures (c-kit, Thy-1, B220, CD11c), confirming that VCAM-1+ cells represent a previously undescribed cell type contained in CP and ILF. In addition to their peripheral pattern of distribution, focused clusters of VCAM-1+ cells were regularly detected within the B cell area of ILF, a pattern reminiscent of VCAM-1+ follicular dendritic cells in a splenic germinal center. We propose that VCAM-1+ cells, which presumably express LTβR, are important for initial interactions with LTα1β2-expressing BM-derived lymphoid progenitor cells in CP and ILF formation, similar to their function in PP development.

FIGURE 6.

CP and ILF contain VCAM-1+ stromal cells. A–D, Wild-type CP. E, CP from CD132-null mouse reconstituted with wild-type BM. F, ILF from CD132-null mice reconstituted with wild-type BM. Horizontal frozen sections of small intestine were stained with the indicated markers and examined by immunofluorescence and confocal microscopy. Alexa Fluor 647-conjugated c-kit staining was pseudocolored red for clarity. In A, VCAM-1 was detected using a purified anti-VCAM-1 primary mAb, biotinylated polyclonal goat anti-rat Ig, and TSA. VCAM-1 staining in B, C, E, and F was detected using a biotinylated anti-VCAM-1 primary mAb followed by TSA. Scale bars: 20 μm in A, C, D, and E; 100 μm in B and F. White circle indicates a CP.

FIGURE 6.

CP and ILF contain VCAM-1+ stromal cells. A–D, Wild-type CP. E, CP from CD132-null mouse reconstituted with wild-type BM. F, ILF from CD132-null mice reconstituted with wild-type BM. Horizontal frozen sections of small intestine were stained with the indicated markers and examined by immunofluorescence and confocal microscopy. Alexa Fluor 647-conjugated c-kit staining was pseudocolored red for clarity. In A, VCAM-1 was detected using a purified anti-VCAM-1 primary mAb, biotinylated polyclonal goat anti-rat Ig, and TSA. VCAM-1 staining in B, C, E, and F was detected using a biotinylated anti-VCAM-1 primary mAb followed by TSA. Scale bars: 20 μm in A, C, D, and E; 100 μm in B and F. White circle indicates a CP.

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The small intestine is an active site of lymphoid organogenesis with at least three morphologically distinct lymphoid aggregates (PP, ILF, and CP) emerging at different times during development. The sequence of cellular and molecular events required for the initiation of PP development has been investigated in detail (19, 21, 22). Many of the same interactions required for PP development are also involved in the early stages of lymph node development (5, 23). The initial steps of both PP and lymph node development can be accounted for by a model in which interactions between lymphoid tissue inducer cells of hemopoietic origin and stromal cells initiate lymphoid organogenesis. Lymphoid tissue inducer cells are CD3CD4+CD45+ cells that also express the t isoform of the RORγ transcription factor (24, 25). The fetal intestinal stromal cells that are engaged by lymphoid tissue inducer cells during the earliest stages of PP development express the VCAM-1 and ICAM-1 adhesion molecules (20). Stimulation of the LTβR on the stromal cells by membrane bound LTα1β2 on the inducer cells is a pivotal initial signal driving organogenesis of PP and lymph nodes (20). LTα expression by hemopoietic cells and LTβR expression by stromal cells are also required for the development of ILF (14). In this study, we have tested the hypothesis that similar LTα- and LTβR-dependent interactions are required to initiate the development of CP.

We did not detect any CP in the small intestine of LTα- and LTβR-deficient mice in this study. While the presence of CP has not been previously analyzed in LTβR-deficient mice, a study from another laboratory reported that CP were present in the small intestine of LTα-deficient mice (4). In addition to finding that LTα-deficient mice we analyzed lacked naturally occurring CP, we also showed that BM-derived precursors from LTα-deficient mice yielded only rare lymphoid aggregates with features of CP when transferred to CD132-null recipients. Because the LTβR has a second functional ligand in addition to LTα1β2 called LIGHT (26), it is possible that LIGHT expression by lymphoid inducer cells and other hemopoietic precursor cells could permit some degree of LTβR-dependent lymphoid aggregate development to proceed in the absence of the LTα gene (27). Environmental conditions such as the load and composition of the commensal enteric flora may affect the degree to which LIGHT can substitute for LTα1β2. The actions of LIGHT on the LTβR have been proposed as a factor contributing to the development of relatively normal mesenteric lymph nodes in LTβ-deficient mice (28). Nevertheless, our studies indicate that LTα1β2 rather than LIGHT is the primary LTβR ligand involved in initiating CP development.

The signal transmitted by LTα1β2 through the LTβR leading to NF-κB activation has been shown previously to go through at least two distinct pathways: a classical NF-κB activation pathway involving IKKα, IKKβ, and IKKγ and an alternate NF-κB activation pathway using NIK, IKKα, p100, and RelB (13). The presence of the mutant NIK in aly/aly mice prevents signaling through the noncanonical NF-κB signaling pathway and blocks development of lymph nodes, PP, and ILF (4, 8). Our analysis of CP development indicates that while LTα and LTβR are both required for CP formation, a normal number of CP that are morphologically indistinguishable from wild-type CP develop in aly/aly mice with a mutant NIK gene. There are at least two possible explanations to account for this finding. One explanation is to postulate that a low level of residual NIK activity in aly/aly mice is sufficient to initiate development of CP, but insufficient to sustain all other types of LTβR-dependent lymphoid development. The mutation in the NIK gene of aly/aly mice is a single nucleotide change resulting in a single amino acid substitution (G855R) (29). The level of the mutant NIK transcript is reduced compared with the wild-type NIK transcript (29, 30), but the mutated NIK kinase domain was shown to retain NF-κB-inducing activity when expressed in vitro by transfection (29). One approach to resolve the question of whether low residual activity of this potential hypomorph allele of NIK can provide sufficient LTβR-dependent signaling to stimulate CP development is to determine whether gene targeted mutant mice in which NIK activity has been completely abrogated have the same types of small intestinal lymphoid aggregates as aly/aly mice (i.e., presence of CP without any ILF). A second explanation to account for the presence of CP in aly/aly mice despite blockade of the alternate NF-κB activation pathway secondary to the NIK mutation is use of a different signaling pathway downstream of the LTβR to trigger NF-κB activation. In the absence of NIK, the classical NF-κB activation pathway remains intact and could transmit an LTβR-dependent signal leading to activation of NF-κB-responsive genes and CP development. Whether the classical NF-κB activation pathway is normally the primary LTβR-initiated signaling pathway leading to CP development or whether both major LTβR signaling pathways are individually sufficient to support CP development cannot be determined from the present set of experimental results.

Stromal cells expressing VCAM-1 play a critical role in LT-dependent development of PP beginning at embryonic day 15.5 in mice (19). Initiation of interactions between lymphoid tissue inducer cells and stromal cells expressing both VCAM-1 and ICAM-1 evenly distributed through the lamina propria of the small intestine leads to clustering of VCAM-1+, ICAM-1+ cells at the site of the future PP (19). In addition, follicular dendritic cells expressing VCAM-1 are required for the normal LT-dependent maturation of germinal centers in B cell follicles in the spleen and lymph nodes (31, 32, 33). In both of these examples, the cells expressing VCAM-1 also express the LTβR. We investigated whether mouse CP and ILF also contain VCAM-1+ cells that could play analagous roles in CP and ILF development. The VCAM-1+ cells concentrated at the periphery of CP are logical candidate cells to transduce the signal supplied by LTα1β2 on the putative inducer cells initiating CP development. VCAM-1+ cells were also consistently detected at the periphery of ILF, as well as in focused clusters within the interior of B cell follicles.

Having established that there is a shared requirement for LTα and LTβR to initiate the postnatal development of CP and ILF in the small intestine and the prenatal development of PP and lymph nodes, further questions about the early development of these small intestinal lymphoid aggregates remain to be addressed in future studies. CP and ILF first appear at approximately the same time postnatally (between 7 and 25 days after birth) (4), but it is not known whether CP and ILF begin development as structures already committed to become CP and ILF or as uncommitted lymphoid aggregates that can differentiate into either CP or ILF depending on the extrinsic environmental signals encountered. Because both CP and ILF can both develop during adult life, steady-state numbers of CP and ILF could be maintained by continuous generation of new CP and ILF to replace structures that disassemble over time. Alternatively, the initial CP and ILF that appear early in the postnatal small intestine may be long-lived structures that normally persist in the same location throughout adult life. Further investigations aimed at identifying at what stage of development intestinal lymphoid aggregates commit to becoming CP or ILF and determining the natural turnover rate of these structures will yield new insights into the factors regulating lymphoid organogenesis in the intestinal microenvironment.

After this paper was submitted for publication, Eberl and Littman (34) reported that mature cryptopatches containing clusters of RORγt+ cells are not present in the small intestine of LTα null mice. This observation made using LTα-null mice heterozygous for a RORγt/EGFP knockin mutation is consistent with the data reported in our study.

We thank Yang-Xin Fu at the University of Chicago for providing small intestinal tissue from LTβR-deficient mice and Kimberly Brown from the Emory University Hospital Histopathology Laboratory for assistance with sectioning of paraffin-embedded tissue.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from the National Institutes of Health (DK64730 to I.R.W. and DK64399 supporting the Imaging Core Facility of the Emory Digestive Diseases Research Development Center), Emory University Research Committee (I.R.W.), and Deutsche Forschungsgemeinschaft (Lu816/1-1 to A.L.).

3

Abbreviations used in this paper: CP, cryptopatch; LT, lymphotoxin; LTβR, lymphotoxin β receptor; ILF, isolated lymphoid follicle; PP, Peyer’s patch; BM; bone marrow; IEL, intraepithelial lymphocyte; TSA, tyramide signal amplification; NIK, NF-κB-inducing kinase; IKK, IκB kinase; NALT, nasal-associated lymphoid tissue.

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